Method for diagnosing and monitoring inflammatory disease progression

ABSTRACT

Methods for diagnosing or monitoring endometriosis in a mammal are provided. The methods include the steps of determining the expression levels of BDNF, glycodelin and optionally ZAG, in a biological sample from the mammal, and determining that the mammal has endometriosis when the biomarker expression levels in the sample are elevated.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application is a continuation-in-part of and claims the benefit ofpriority to U.S. Utility application Ser. No. 15/094,086, filed Apr. 8,2016, which claims the benefit of priority, under 35 U.S.C. § 120, fromthe US designation of International Application No. PCT/CA2014/000742,filed on Oct. 10, 2014, which claims benefit of priority from U.S.Provisional Application Ser. No. 61/889,085, filed on Oct. 10, 2013, theentire content of each of which is incorporated herein by reference inits entirety for all purposes.

FIELD OF THE INVENTION

The present invention provides compositions and/or methods for diagnosisor assessment of progression of inflammatory diseases, in particular,endometriosis.

BACKGROUND OF THE INVENTION

Neurotrophins are a family of soluble, small molecular weight proteinsthat act in the nervous system to promote neuronal development,differentiation, growth, and maintenance. The neurotrophin signallingnetwork is complex. Neurotrophins can be translated as pro-proteins andcleaved into their active forms, or they can induce signalling cascadesin their pro-form. Generally, the two forms have opposing functions. Theneurotrophin family comprises four ligands, brain-derived neurotrophicfactor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NTF3), andneurotrophin 4 (NTF4), and four receptors: neurotrophic tyrosinereceptor kinase (NTRK) 1, NTRK2, NTRK3, and the nerve growth factorreceptor (NGFR). Although all four neurotrophins bind to NGFR withsimilar affinities, and their pro-protein forms have been shown to bindto this receptor as well, they are more selective in binding the NTRKs.NGF binds to NTRK1, BDNF and NTF4 bind to NTRK2, and NTF3 binds toNTRK3, each with high affinity. Another lesser known neurotrophinco-receptor, sortilin (SORT1), has been shown to interact withpro-neurotrophins in the brain and to control their release in either aconstituent or activity-dependent manner. SORT1 is also involved in anelaborate intracellular trafficking network directing proteins tovarious fates: cell surface expression, secretion, endocytosis, ortransport within the cell. However, the regulation and expression ofthis complex signalling network in the uterus remains unexplored.

Although mainly recognized for their supportive function within thenervous system, BDNF and its high affinity receptor NTRK2 have beenshown to participate in ovarian development, follicular development,oocyte survival, endometrial stem cell neurogenesis, and normalplacental development. The interaction between BDNF and NTRK2 is notonly capable of inducing neuronal development, differentiation, growth,and maintenance; activation of the BDNF-NTRK2 pathway has beendemonstrated to induce angiogenesis, cellular proliferation, adhesion,and resistance to apoptosis. Each of these pathways is inextricablylinked to reproduction, however the mechanisms regulating the uterineexpression of BDNF, NTRK2, NGFR, and SORT1 remain unknown.

Thus, it would be desirable to better understand neurotrophin regulationin the mammalian uterus, and to develop methods to recognize one or morepathologies associated with a neurotrophin.

SUMMARY OF THE INVENTION

It has now been determined that elevated expression levels of BDNFcombined with one or more additional biomarkers, such as full-lengthNtrk2 receptor, glycodelin and optionally, zinc-alpha-2-glycoprotein(ZAG), in a biological sample from a mammal is indicative ofendometriosis.

Thus, in one aspect, a method of diagnosing endometriosis in a mammal isprovided comprising the steps of: determining the expression level ofBDNF in a biological sample from the mammal and comparing the BDNF levelto a control BDNF level; determining the expression level of full-lengthNtrk2 in the biological sample and comparing the Ntrk2 level to acontrol Ntrk2 level; and diagnosing the mammal with endometriosis whenthe BDNF level and Ntrk2 level are both elevated by at least 10% ascompared with the control levels.

In another aspect, a method of diagnosing endometriosis in a mammal isprovided comprising the steps of: determining the expression levels ofBDNF, glycodelin, and optionally ZAG, in a biological sample from themammal and comparing the level of each to a pre-determined levelassociated with endometriosis; and diagnosing the mammal withendometriosis when the levels of BDNF, glycodelin and optionally ZAG areeach elevated to the predetermined level associated with endometriosis.

In another aspect, a method of monitoring a mammal following treatmentfor endometriosis is provided comprising: determining the expressionlevel of a biomarker selected from BDNF, or glycodelin in a biologicalsample from the mammal, comparing the biomarker level to a pre-treatmentlevel, and determining that the mammal is responding to treatment if thebiomarker level is reduced by at least 10% as compared to thepretreatment biomarker level.

In a further aspect of the invention, a kit is provided comprising aBDNF-specific reactant and i) a glycodelin-specific reactant andoptionally a ZAG-specific reactant, or ii) a full-length Ntrk2-specificreactant, and further optionally, instructions for use to detectendometriosis in a mammal.

In a further aspect, a method of diagnosing inflammatory disease in amammal is provided. The method comprises determining the expressionlevel of BDNF in a biological sample from the mammal and comparing theBDNF level to a control BDNF level to determine if the BDNF level iselevated in comparison to the BDNF baseline level, wherein an elevatedBDNF level is indicative of inflammatory disease in the mammal.

These and other aspects of the invention are described herein byreference to the description and figures as follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 graphically illustrates that circulating concentration of BDNF ishigher in the plasma of women with endometriosis vs. a controlpopulation;

FIG. 2 graphically illustrates that total plasma BDNF concentration issignificantly higher in women at any stage of endometriosis vs.controls;

FIG. 3 graphically demonstrates that plasma total BDNF concentration issimilar across the menstrual cycle;

FIG. 4 compares plasma BDNF concentration in women with endometriosisprior to treatment (untreated), and subsequent to treatment (treated);

FIG. 5 illustrates the relationship between plasma BDNF concentrationsand pain scores in mammals with untreated endometriosis;

FIG. 6 shows the results of Western blot analysis of human endometriumfrom healthy women illustrating that pro-BDNF is the dominant formpresent (A), and that truncated Ntrk2 is the dominant isoform present(B);

FIG. 7 shows a Western blot analysis of endometrium obtained from womenwith endometriosis vs. healthy controls showing that the full-length(FL) variant of Ntrk2 is overexpressed in endometriosis;

FIG. 8 illustrates BDNF transcript expression in the murine uterus inmice receiving saline (Control (n=4)), estradiol primed then estradiol(E2 (n=6)), estradiol primed then progesterone (P4 (n=6)), estradiolprimed then estradiol+progesterone (E₂+P₄ (n=6)), or estradiol primedthen saline (Saline (n=4));

FIG. 9 illustrates the amino acid sequences of human (A), mouse (B) andrat (C) mBDNF, and of human (D), mouse (E) and rat (F) full-lengthNtrk2;

FIG. 10 illustrates the nucleic acid sequence of human (A), mouse (B)and rat (C) BDNF transcripts, and human (D), mouse (E) and rat (F) Ntrk2transcripts;

FIG. 11 illustrates circulating concentrations of ZAG, glycodelin, BDNF,and CA-125 between untreated cases (n=35-60) and controls (n=12-26) withROC curves. Circulating glycodelin (A) was significantly elevated(p=0.041) in untreated cases compared to controls. Circulating ZAG (B)tended to be elevated (p=0.086) in untreated cases compared to controls.Circulating BDNF (C) was significantly higher (p=0.0008) in untreatedcases compared to controls while circulating concentrations of CA125 (D)did not reach statistical significance (p=0.626). Glycodelin, ZAG, BDNF,and CA-125 produced ROC curves with an AUC of 0.70 (p=0.040), 0.66(p=0.085), 0.73 (p<0.001), and 0.47 (p=0.622), respectively.Statistically significant differences are denoted by an asterisk (*)above the graph (* p<0.05; *** p<0.001). Whiskers on the box plotsrepresent the 5th and 95th percentiles, while the lower limit of the boxlower quartile and the upper limit is the upper quartile. The linewithin the box is the median of the data. Normally distributed data areportrayed as an aligned dot plot with error bars representing standarddeviation from the mean;

FIG. 12 illustrates that a biomarker decision tree utilizing defaultCART analysis parameters achieves a sensitivity of 76.9% and aspecificity of 93.3%. Parent nodes are outlined by bold blue rectanglesand terminal nodes are outlined by red rectangles. The class assignmentof patients in each node is shown under the node number. Class 0 is thecontrol group, and class 1 is the endometriosis group. Bars give agraphical representation of the proportion of patients from each groupassigned to that node. Splitting variables are shown above a node, withthe cut-off value for the split shown above the child node in gray.N=number of study participants;

FIG. 13 illustrates that a biomarker decision tree utilizing CARTanalysis parameters optimized for sensitivity achieves a sensitivity of89.2% and a specificity of 70.0%. Parent nodes are outlined by bold bluerectangles and terminal nodes are outlined by red rectangles. The classassignment of patients in each node is shown under the node number.Class 0 is the control group, and class 1 is the endometriosis group.Bars give a graphical representation of the proportion of patients fromeach group assigned to that node. Splitting variables are shown above anode, with the cut-off value for the split shown above the child node ingray. N=number of study participants;

FIG. 14 illustrates the amino acid sequences of human isoforms ofglycodelin (A, B and C), and human transcript variants of humanglycodelin (D, E and F); and

FIG. 15 illustrates the amino acid sequences of human (A) and mouse (B)ZAG, and mRNA sequences of human (C) and mouse (D) ZAG.

FIG. 16 is a schematic illustrating: a) APTES(3-aminopropyltiriethoxysilane) treated polystyrene (PS) substrate withporous gold immobilization surface; b) thiol bonding immobilizes theprimary linker cystamine followed by an easily reactive secondaryglutaraldehyde linker to create a self-assembled monolayer of aldehydegroups; c) anti-BDNF (Brain Derived Neurotrophic Factor) monoclonalantibody (mAb) is immobilized via the secondary linker to assemble theelectrochemical detection device; d) unreacted aldehyde groups areblocked using 5% (w/v) Bovine Serum Albumin (BSA); e) the targetanalyte, BDNF protein, will attach to its mAb, and the redox reportersystem of [Fe(CN)₆]^(3−/4−) is used to electrochemically detect thepresence of protein; and f) Differential Pulse Voltammetry graphillustrating how the binding of BDNF to the antibody inhibits theinterfacial electron transfer reaction to take place thereforedecreasing current signal.

FIG. 17 graphically compares the sensitivities of planar (grey bar) andporous (black bar) immunosensors when a concentration of 1 ng/mL BDNF inPBS is used.

FIG. 18 graphically illustrates the detection limit of sensors in aelectrochemical solution of 2.5 mM [Fe(CN)₆]^(3−/4−) with variousconcentrations of BDNF in PBS.

FIG. 19 illustrates differential pulse voltammograms that show thedifference in electrochemical signal before and after the addition oftarget analyte at various concentrations.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, a method of diagnosing endometriosis in a mammal isprovided comprising the steps of: determining the expression level ofBDNF in a biological sample from the mammal and comparing the level to acontrol BDNF baseline level; determining the expression level offull-length Ntrk2 in the biological sample from the mammal and comparingthe Ntrk2 level to a control Ntrk2 baseline level; diagnosing the mammalwith endometriosis when the BDNF level and Ntrk2 level are both elevatedby at least 10% as compared with their baseline levels.

In another aspect, a method of diagnosing endometriosis in a mammal isprovided comprising the steps of: determining the expression levels ofBDNF, glycodelin, and optionally ZAG, in a biological sample from themammal and comparing the level of each to a pre-determined levelassociated with endometriosis; and diagnosing the mammal withendometriosis when the levels of BDNF, glycodelin and optionally ZAG areeach elevated to the predetermined level associated with endometriosis.

Brain-derived neurotrophic factor, referred to herein as BDNF, is asecreted protein that supports growth and survival of neurons. As usedherein, BDNF encompasses mammalian BDNF, including human andfunctionally equivalent variants thereof such as non-human BDNF, andisoforms or other variants of human and non-human BDNF, includingpro-BDNF and mBDNF. Functionally equivalent BDNF variants are variantsthat incorporate alterations, such as, but not limited to, amino aciddeletions, additions or substitutions, which do not significantlyadversely affect BDNF activity. Post-translationally modified BNDF isreferred to as mature BDNF or mBDNF. Amino acid sequences for mBDNF areknown and readily accessible at sequence databases, such as GenBank, byreference to nucleotide accession nos., e.g. human mBDNF (accession no.KC855559), mouse mBDNF (accession no. KC855560), rat mBDNF (accessionno. KC855561), pig mBDNF (accession no. KC855563) and horse mBDNF(accession no. KC855562). mBDNF amino acid sequences are illustrated inFIG. 9, and nucleic acid encoding sequences are shown in FIG. 10.

Neurotrophic tyrosine kinase, receptor, type 2 (Ntrk2), also known asTrkB receptor, TrkB tyrosine kinase or BDNF/NT-3 growth factor receptor,is a BDNF receptor. As used herein, Ntrk2 encompasses full-lengthmammalian Ntrk2, including human and functionally equivalent variantsthereof such as non-human Ntrk2. Functionally equivalent variants offull-length Ntrk2 encompass full-length Ntrk2 which may incorporatealterations, such as, but not limited to, minor amino acid alternationssuch as deletions, additions or substitutions, e.g. involving 1 or 2amino acid residues, which do not significantly adversely affect Ntrk2activity, such as BDNF binding. Amino acid sequences of various forms offull-length Ntrk2 are known and readily accessible at sequencedatabases, such as GenBank, by reference to nucleotide accession nos.,e.g. human Ntrk2 (KC855566), mouse Ntrk2 (KC855567), rat Ntrk2(KC855568) and horse Ntrk2 (KC855569). Ntkr2 amino acid sequences areillustrated in FIG. 9, and nucleic acid encoding sequences are shown inFIG. 10.

Glycodelin, also known as progestagen-associated endometrial protein(PAEP) or pregnancy-associated endometrial alpha-2 globulin, is aprotein that in humans is encoded by the PAEP gene. As used herein,glycodelin encompasses mammalian glycodelin, including human andfunctionally equivalent variants thereof such as non-human glycodelin,and isoforms or other variants of human and non-human glycodelin, whichessentially retain the function of the parent protein. Functionallyequivalent glycodelin variants are variants that incorporatealterations, such as, but not limited to, amino acid deletions,additions or substitutions, which do not significantly adversely affectactivity. Amino acid sequences for glycodelin are known and readilyaccessible on sequence databases, such as NCBI, by reference toaccession nos. e.g. human glycodelin (accession no. NP_001018058(Isoform 2 precursor); and NP_001018059 (isoform 1 precursor)), as shownin FIG. 14A, as well as nucleotide sequences, transcript variants 1 and2, which encode isoform 1, and transcript variant 3 which encodesisoform 2 (accession nos. NM_001018049, NM_002571 and NM_001018048,respectively) as shown in FIG. 14B.

Zinc-alpha-2-glycoprotein (ZAG) is a protein that in humans is encodedby the AZGP1 gene. As used herein, ZAG encompasses full-length mammalianZAG, including human and functionally equivalent variants thereof suchas non-human ZAG. Functionally equivalent variants of full-length ZAGencompass full-length Ntrk2 which may incorporate alterations, such as,but not limited to, minor amino acid alternations such as deletions,additions or substitutions, e.g. involving 1 or 2 amino acid residues,which do not significantly adversely affect Ntrk2 activity, such as BDNFbinding. Amino acid sequences of various forms of ZAG are known andreadily accessible from sequence databases, such as NCBI, by referenceto accession nos., e.g. human ZAG (NP_001176) and mouse ZAG (NP_038506),as well as transcript sequences for human ZAG (NM_001185) and mouse ZAG(NM_013478) as shown in FIG. 15.

To conduct the present method, a suitable biological sample(s) isobtained from a female mammal. The term “biological sample” is meant toencompass any mammalian fluid or tissue sample that may contain nucleicacid encoding a target biomarker gene, or that may contain the targetbiomarker protein (such as BDNF, Ntrk2, glycodelin and/or ZAG protein ornucleic acid). Suitable biological samples include, for example, blood(including menses), serum, plasma, urine, peritoneal fluid or biopsiedendometrial tissue. Any of these samples may be obtained from the mammalin a manner well-established in the art. The term “mammal” is usedherein to refer to both human and non-human mammals including domesticanimals, e.g. cats, dogs and the like, livestock and undomesticatedanimals.

Once a suitable biomarker-containing biological sample is obtained, itis analyzed to determine the expression level of selected biomarkers inthe sample, either at the transcript level or protein level. As one ofskill in the art will appreciate, the expression level of each biomarkermay be determined using one of several techniques established in theart, including methods of quantifying nucleic acid encoding the targetbiomarker, such as PCR-based techniques, microarrays, gene expressionsystem, and Northern or Southern blotting techniques, or methods ofquantifying protein biomarker, such as immunological or activity assay,Western blotting, or mass spectrometry. With respect to BDNF, it is thelevel of mBDNF that is related to endometriosis; however, total BDNFdoes reflect changes in mBDNF. Thus, depending on the biological sampleused, either the expression level of total BDNF may be determined, or,if possible in the sample obtained, the expression level of mBDNF may bespecifically determined.

In one embodiment, the expression levels of biomarkers (e.g. BDNF,Ntrk2, glycodelin or ZAG) in a biological sample from a mammal may bedetermined based on the levels of nucleic acid (i.e. DNA or mRNAtranscript) encoding the target protein biomarker in the biologicalsample. Methods of determining DNA or mRNA levels are known in the art,and include, for example, PCR-based techniques (such as RT-PCR), andNorthern or Southern blotting techniques which generally include theapplication of gel electrophoresis to isolate the target nucleic acid,followed by hybridization with specific labeled probes. Probes for usein these methods can be readily designed based on the known sequences ofgenes encoding the protein biomarker, as well as the known amino acidsequence of the target biomarker, and may comprise about 15-40nucleotides, for example, 20-35 nucleotides. Probes that target mBDNFare generally suitable for use in the present method. Such probes woulddetect total BDNF in a sample. For Ntrk2, probes that target full-lengthNkrt2 are generally suitable to detect Ntrk2. Examples of BDNF probesinclude GAGCTGAGCGTGTGTGACAG (forward) (SEQ ID NO: 9) andCTTATGAATCGCCAGCCAAT (reverse) (SEQ ID NO: 10), and examples of Ntrk2probes include CAATTGTGGTTTGCCATCTG (forward) (SEQ ID NO: 11) andTGCAAAATGCACAGTGAGGT (reverse) (SEQ ID NO: 12). Suitable labels for useare well-known, and include, for example, fluorescent, chemiluminescentand radioactive labels. Probes for glycodelin and ZAG may readily bedetermined based on their known gene sequences, including the mRNAsequences provided herein.

A preferred assay method to measure biomarker transcript abundanceincludes using the NanoString nCounter gene expression system. Thesystem utilizes a pair of probes, namely, a capture probe and a reporterprobe, each comprising a 35- to 50-base sequence complementary to thebiomarker transcript. The capture probe additionally includes a shortcommon sequence coupled to an immobilization tag, e.g. an affinity tagthat allows the complex to be immobilized for data collection. Thereporter probe additionally includes a detectable signal or label, e.g.is coupled to a color-coded tag. Following hybridization, excess probesare removed from the sample, and hybridized probe/target complexes arealigned and immobilized via the affinity or other tag in a cartridge.The samples are then analyzed, for example using a digital analyzer orother processor adapted for this purpose. Generally, the color-coded tagon each transcript is counted and tabulated for each target transcriptto yield the expression level of each transcript on the sample.

In other embodiments, the expression level of protein, mBDNF,full-length Ntrk2, glycodelin or ZAG, in a sample may be measured byimmunoassay using an antibody specific to the target protein. As above,the antibody is bound to the target protein and bound antibody isquantified by measuring a detectable marker which may be linked to theantibody or other component of the assay, or which may be generatedduring the assay. Detectable markers may include radioactive,fluorescent, phosphorescent and luminescent (e.g. chemiluminescent orbioluminescent) compounds, dyes, particles such as colloidal gold andenzyme labels.

The term “antibody” is used herein to refer to monoclonal or polyclonalantibodies, or antigen-binding fragments thereof, e.g. an antibodyfragment that retains specific binding affinity for the targetbiomarker. Antibodies to the target biomarkers are generallycommercially available. For example, BDNF antibodies to various BDNFimmunogens, including internal, and N- and C-terminal, are commerciallyavailable, for example, from Sigma Alderich, Santa Cruz Biotechnologyand AbCam, while Nkrt2 antibodies are commercially available from, forexample, AbCam, R&D Systems and Origene Technologies. Antibodiestargeting glycodelin and ZAG are similarly commercially available fromAbCam, LifeSpan BioSciences, R&D Systems, Santa Cruz Biotechnology andothers. As one of skill in the art will appreciate, antibodies to thetarget proteins may also be raised using techniques conventional in theart. For example, antibodies may be made by injecting a host animal,e.g. a mouse or rabbit, with the antigen (target protein or immunogenicfragment thereof), and then isolating antibody from a biological sampletaken from the host animal.

Different types of immunoassay may be used to determine the expressionlevel of target proteins, including indirect immunoassay in which theprotein is non-specifically immobilized on a surface; sandwichimmunoassay in which the protein is specifically immobilized on asurface by linkage to a capture antibody bound to the surface;competitive binding immunoassay in which a sample is first combined witha known quantity of antibody to bind the target protein in the sample,and then the sample is exposed to immobilized target protein whichcompetes with the sample to bind any unbound antibody. To theimmobilized protein/antibody is added a detectably-labeled secondaryantibody that detects the amount of immobilized primary antibody,thereby revealing the inverse of the amount of target protein in thesample.

A preferred immunoassay for use to determine expression levels of targetprotein in a sample is an ELISA (Enzyme Linked ImmunoSorbent Assay) orEnzyme ImmunoAssay (EIA). To determine the level or concentration of thetarget protein using ELISA, the target to be analyzed is generallyimmobilized, for example, on a solid adherent support, such as amicrotiter plate, polystyrene beads, nitrocellulose, cellulose acetate,glass fibers and other suitable porous polymers, which is pretreatedwith an appropriate ligand for the target, and then complexed with aspecific reactant or ligand such as an antibody which is itself linked(either before or following formation of the complex) to an indicator,such as an enzyme. Detection may then be accomplished by incubating thisenzyme-complex with a substrate for the enzyme that yields a detectableproduct. The indicator may be linked directly to the reactant (e.g.antibody) or may be linked via another entity, such as a secondaryantibody that recognizes the first or primary antibody. Alternatively,the linker may be a protein such as streptavidin if the primary antibodyis biotin-labeled. Examples of suitable enzymes for use as an indicatorinclude, but are not limited to, horseradish peroxidase (HRP), alkalinephosphatase (AP), ß-galactosidase, acetylcholinesterase and catalase. Alarge selection of substrates is available for performing the ELISA withthese indicator enzymes. As one of skill in the art will appreciate, thesubstrate will vary with the enzyme utilized. Useful substrates alsodepend on the level of detection required and the detectioninstrumentation used, e.g. spectrophotometer, fluorometer orluminometer. Substrates for HRP include 3,3′,5,5′-Tetramethylbenzidine(TMB), 3,3′-Diaminobenzidine (DAB) and2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS).Substrates for AP include para-Nitrophenylphosphates. Substrates forß-galactosidase include β-galactosides; the substrate foracetylcholinesterase is acetylcholine, and the substrate for catalase ishydrogen peroxide.

As will be appreciated by one of skill in the art, assay methods whichtarget the activity of a target protein may also be utilized todetermine the expression level thereof in a sample. In this regard,suitable assays would be known to the skilled person, including forexample, an mBDNF-Nkrt2 binding assay.

The expression level of the selected biomarkers mBDNF and Nkrt2, ormBDNF, glycodelin and optionally ZAG, in a given sample may be analyzedindividually or together using, for example, biochip array technology.Generally, biochip arrays provide a means to simultaneously determinethe level of multiple biomarkers in a given sample. These arrays mayutilize ELISA technology and, thus, the biochip may be modified toincorporate capture antibodies for each target at pre-defined sites onthe surface.

Once the expression level of selected biomarkers in a biological sampleof a mammal has been determined, these expression levels are compared tocontrol expression levels, i.e. the expression level of selectedbiomarkers from BDNF, Ntrk2, glycodelin and ZAG, in a healthy control,i.e. a mammal that does not have endometriosis. Alternatively, the levelof the selected biomarkers may be compared to the expression level of a“housekeeping gene”. The term “housekeeping gene” as used herein ismeant to refer to a gene that encodes a protein product that is notconnected to, involved in or required for processes specific toendometriosis, and thus, exhibits a fixed expression level in mammalswith and without endometriosis. Examples of suitable housekeeping genesinclude, but are not limited to, genes encoding ACTB (Beta-actin), GAPDH(Glyceraldehyde 3-phosphate dehydrogenase), RPLP0 (60S acidic ribosomalprotein P0), GUSB (beta-glucuronidase), and TFRC (transferring receptor1). In a comparison of the expression levels of target biomarkers tohousekeeping genes, a determination of an increase in transcriptabundance or expression of the selected biomarker relative to that ofthe housekeeping gene is indicative of endometriosis.

The level of expression (or concentration) that would be considered torepresent an increased or elevated expression level of the selectedbiomarkers that is associated with endometriosis in accordance with thepresent method may be determined relative to levels of biomarker in ahealthy control sample, or relative to the expression of one or morehousekeeping genes. In one embodiment, a reproduceable statisticallysignificant increase in the expression of a biomarker, for example, anincrease of at least about 5%, preferably, at least about 10%, 20%, 30%,40% or 50% or greater, in comparison to the expression levels in acontrol, or in comparison to the expression level of a housekeepinggene, is considered to be elevated expression that is relevant withrespect to a diagnosis of endometriosis. Generally, a plasma BDNF levelin the range of about 100-500 pg/ml is considered to be normal, whileplasma BDNF levels higher than this, e.g. by about 10-50% or greater,are indicative of endometriosis, e.g. for example, plasma BDNF levels of800 pg/ml or greater are indicative of endometriosis. Generally, serumconcentrations of glycodelin in the range of 5 to 31 ng/ml is consideredto be normal, and concentrations greater than 39 ng/ml are considered tobe indicative of endometriosis. For ZAG, circulating concentrations inthe range of 41 to 65 pg/ml are regarded as normal, while serumconcentrations greater than 92 pg/ml are considered to be indicative ofendometriosis. As one of skill in the art will appreciate, thedifference in the level of biomarker expression as compared toexpression of the housekeeping gene(s) may vary with the methodologyemployed to quantify and analyze nucleic acid and/or protein expression.

The present invention also provides a method of diagnosing the stage ofendometriosis. Levels of BDNF exhibit a greater increase in comparisonto normal controls at stage I-II of endometriosis than at stage whilelevels of glycodelin exhibit a greater increase in comparison to normalcontrols at stage III-IV of endometriosis than at stage I-II. Thus,levels of BDNF which are greater than 30%, and preferably, 40-50%greater than control levels, are indicative of stage I-II endometriosis,and levels of glycodelin which are greater than 30%, and preferably,40-50% greater than control levels, are indicative of stage III-IVendometriosis.

Once a mammal has been diagnosed with endometriosis, the mammal can thenbe appropriately treated. In mild cases, the appropriate treatment maybe administration of a pain medication, such as nonsteroidalanti-inflammatory drugs (NSAIDs), e.g. ibuprofen or naproxen, to addresspainful cramps. Alternatively, hormone therapy may be utilized toaddress the pain mild to moderate endometriosis, including, hormonalcontraceptives (birth control pills, patches and vaginal rings);gonadotropin-releasing hormone (Gn-RH) agonists and antagonists to blockthe production of ovarian-stimulating hormones, lowering estrogen levelsand preventing menstruation, optionally in combination with a low doseof estrogen or progestin to decrease menopausal side effects; progestintherapy, e.g. such as an intrauterine device (Mirena™), contraceptiveimplant or contraceptive injection (Depo-Provera™); and steroidtreatment (e.g. danazol) to suppress the growth of the endometrium. Forsevere endometriosis, treatment by surgery is appropriate.

In another aspect, a method to monitor response by a mammal to treatmentfor endometriosis, including surgical or drug therapy (e.g. hormonetherapy), is also provided. The method of monitoring a mammal followingtreatment of endometriosis comprises: determining the expression levelof a selected biomarker, e.g. BDNF or glycodelin, in a sample from themammal and comparing the level to a pre-treatment biomarker expressionlevel to determine if the biomarker level has decreased in comparison tothe pre-treatment biomarker level. A significant decrease, i.e. adecrease of at least about 10% or greater, preferably at least about 20%or greater, e.g. 30-50% or greater in the biomarker BDNF level comparedto the pre-treatment level indicates that the mammal is responding tothe treatment.

Disease recurrence may also be monitored in a mammal previouslysuccessfully treated for endometriosis using a method in accordance withthe invention. A method as used to diagnose endometriosis in a firstinstance would be applicable. In particular, biomarker levels, e.g. BDNFexpression levels, either total BDNF or mBDNF, and Ntrk2 expressionlevels, or BDNF, glycodelin and optionally ZAG levels, are determined ina relevant biological sample from the mammal as described. It is thendetermined whether or the biomarker levels represent a significantincrease in comparison to control values, or in comparison to the levelof a selected housekeeping gene, wherein a significant increase (e.g.10-50% or greater) in the biomarker levels is indicative of diseaserecurrence.

In a further embodiment of the invention, a kit for use in detectingendometriosis is provided comprising reactants for the specificidentification of selected biomarkers, including a BDNF-specificreactant and a full-length Ntrk2-specific reactant, or a BDNF-specificreactant, a glycodelin-specific reactant and optionally a ZAG-specificreactant. Instructions for use in methods of diagnosing and/ormonitoring disease recurrence, disease progression or treatment ofendometriosis in a mammal may also be provided. As set out above,biomarker-specific reactants may include nucleic acid probes orantibodies based on the known nucleic acid and amino acid sequences ofthe selected biomarkers. A substrate for each biomarker may also be usedas a reactant. The reactants may be associated with an indicator (e.g.an enzyme, such as horseradish peroxidase (HRP), alkaline phosphatase(AP), ß-galactosidase, acetylcholinesterase and catalase) such that theinteraction of the reactant with BDNF and Ntrk2 yields a product orsignal, releases the enzyme that is readily detectable and indicative ofBDNF and Ntrk2 in the biological sample.

The kit may be provided in the form of a biochip which incorporates atleast the selected biomarker-specific reactants, such as a BDNF-specificreactant (or mBDNF reactant) and/or a full-length Ntrk2-specificreactant, or BDNF-, glycodelin- and optionally ZAG-specific reactants,or all of the above, at pre-defined sites on the surface thereof. Thereactants are each associated with an indicator such that in thepresence of the targeted biomarker, a detectable product or signal isreleased, as above. The biochip may be adapted for use with a bloodsample, e.g. from a finger prick, or a menses sample.

In another embodiment, a biochip adapted for the electrochemicaldetection of circulating target biomarkers is provided. One or morebiomarker-specific reactants, e.g. a BDNF-specific reactant, such as anantibody, and optionally, an Ntrk2-specific reactant, or BDNF-,glycodelin- and optionally ZAG-specific reactants, is/are bonded tocircuits, e.g. an electrode, in a silicone microchip. When a targetbiomarker such as BDNF from a sample binds to its specific reactant, italters the voltage potential measured across the probe resulting in ameasurable electrical output that is detectable by transducers in thedevice and which is proportional to the concentration of BDNF in thesample.

Thus, in one embodiment, a device comprising a 3 electrode system isprovided. The device comprises one or more working electrodes adapted tobind with target analyte (e.g. BDNF, full-length Ntrk2, glycodelin, andoptionally, ZAG), i.e. the working electrode has bound theretobiomarker-specific reactant that binds to the target analyte(s); areference electrode with a known reduction potential (acts as referencein measuring and controlling the working electrode's potential and at nopoint does it pass any current); and an auxiliary or counter electrodethat passes all the current needed to balance the current observed atthe working electrode. In use, the electrode system, submerged inelectrolyte comprising a redox reporter, produces an electrochemicalsignal which decreases in the presence of target analyte (which blocksaccess of the redox reporter to the electrode surface). The degree ofsignal change correlates with the concentration level of the targetanalyte. Thus, the device is useful to detect the levels of targetbiomarkers, to determine whether the biomarker levels are associatedwith endometriosis.

Measurement of plasma concentrations of total and/or mBDNF ispotentially valuable as a method to measure non-specific inflammation ina mammal that may be useful to prompt further investigation into thecause thereof. Thus, in another aspect, a method of diagnosinginflammation-causing disease in a mammal is provided. The methodcomprises determining the level of BDNF in a BDNF sample from the mammaland comparing the level to a control BDNF baseline level to determine ifthe BDNF level is elevated in comparison to the BDNF baseline level,wherein an elevated BDNF level is indicative of inflammation-causingdisease in the mammal. Inflammation-causing disease may include, forexample, cancer such as ovarian cancer and other endocrine tumors,lupus, Crohn's disease, ulcerative colitis, polycystic ovarian syndromeand periodontal disease.

Embodiments of the invention are described by reference to the followingspecific examples which are not to be construed as limiting.

Example 1—BDNF Determination by Immunoassay Materials and Methods

Study Participants. Women undergoing surgery for endometriosis (cases,N=76) or other benign gynaecological surgeries (symptomatic controls,N=20) were recruited prospectively. Women with no history of pelvicpain, who were not undergoing surgery were also recruited (asymptomaticcontrols, N=18). Study participants completed demographics andgynecological history questionnaires. Menstrual cycle length, date oflast menstruation, and pelvic pain assessed on a 5 question, 5-pointvisual analog scale was recorded for each participant. Women whounderwent laparoscopic surgery were categorized as a case or symptomaticcontrol by a gynaecological surgeon, and the diagnoses were laterconfirmed with pathology reports. The stage of endometriosis wasdetermined during surgery according to the revised Classification of theAmerican Society of Reproductive Medicine. All study participantscompleted written informed consents and the study was approved by theHamilton Health Sciences Integrated Research Ethics Board, McMasterUniversity (IRB #06-064, and 12-083-T).

A trained research nurse collected peripheral blood from the cubitalvein from each participant in plasma separator tubes (BD Canada,Mississauga, ON, Canada). Blood was placed on ice, transported to thelaboratory, and processed according to established Standard OperatingProcedures (SOPs: MAC-OG-RBF-001 to MAC-OG-RBF-006). Immediately afterplasma separation, plasma was divided into 1.8 mL cryovials(Sigma-Aldrich Chemical Company, St. Louis. Mo.) and frozen at −80° C.until required for assay.

Exclusions. Individuals unable to provide consent, or under the age of18 were excluded from the study. Women were also excluded from the studyif they had a diagnosis of adenomyosis (4.4%), polycystic ovary syndrome(0.9%), or if the pathological findings did not correlate with theclinical impression (4.4%). Plasma samples were excluded from the studyif they were hemolyzed (13.2%).

BDNF Assay. Plasma samples were thawed at room temperature andcirculating BDNF was quantified in triplicate using the BDNF Emaximmunoassay ELISA (Promega, Madison, Wis., USA), following themanufacturer's protocol. Briefly, 96 well NUNC maxisorp plates werecoated with anti-human BDNF antibody (provided with the kit) overnight.They were blocked the following morning using the block and samplebuffer provided in the kit. Freshly thawed plasma samples were diluted1:10 with sample buffer provided in the kit. Following the BDNF ELISA,the absorbance was read at 450 nm within 30 minutes using the BiotekSynergy spectrophotometer (Fisher Scientific, Ottawa, ON, Canada). BDNFconcentration and % CV of the triplicates were calculated by the BiotekSynergy software.

Data and Statistical Analysis. The intra-sample variation (triplicates)did not exceed 15% (% CV<15) in any plasma sample. A Grubb's test(http://graphpad.com/quickcalcs/Grubbs1.cfm) was used to identifystatistical outliers (2.6%) which were omitted from analysis and valuesfound to be non-detectable by ELISA (1.8%). Data were compared byt-test, one-way ANOVA, or linear regression (SigmaStat 3.5 SystatSoftware Inc., Chicago, Ill., USA). A P value of <0.05 was consideredsignificant. Data are presented as box plot with lines representing the25^(th), 50th, and 75th percentiles.

Results:

Circulating concentrations of BDNF were found to be higher in the plasmaof women with endometriosis vs. symptomatic and asymptomatic controls asshown in FIG. 1. The data included only women not receiving hormonetreatment for endometriosis. The control group was composed of 36%symptomatic and 64% asymptomatic mammals. The endometriosis groupincluded mammals in endometriosis stages I and II (15%) and stages IIIand IV (85%). Comparisons were made by t-test. Mean total plasma BDNFconcentrations were significantly higher in women with stage I and IIendometriosis, and with stages III and IV endometriosis vs. controls(see FIG. 2). Data were compared by one-way ANOVA and appropriateposthoc comparison test.

A comparison of plasma total BDNF concentrations across the menstrualcycle (menses, follicular and luteal) indicated that the concentrationwas similar during the entire cycle (see FIG. 3).

Plasma BDNF concentrations were compared in controls (women withoutendometriosis), women with endometriosis prior to ovarian suppressiontreatment (untreated), and women with endometriosis who were beingtreated for their disease with ovarian suppressing hormone treatments(oral contraceptives or Lupron). These results showed that treatment ofendometriosis resulted in a decrease in plasma BDNF to levels comparableto control levels (FIG. 4). Data were compared by one-way ANOVA andappropriate posthoc test.

A comparison of plasma BDNF concentrations against pain was alsoconducted. BDNF plasma concentrations were found to be positivelycorrelated with pain scores, which is a primary presenting complaint ofmammals with endometriosis. Linear Regression performed using pain asthe dependent variable and plasma BDNF concentration as the independentvariable.

Example 2—mBDNF and Ntrk2 Expression Linked to Endometriosis

Western blot analysis of BDNF and Ntrk2 from human endometrium ofhealthy women was conducted. Extracted protein (60 mg) from humanendometrium was run on a 4-20% gradient gel (Thermo-Scientific) at 150 Vfor 50 minutes. Protein was transferred to PVDF membrane (VWRInternational, Mississauga, ON, Canada) at 40 V for 90 minutes. Blotswere blocked for 1 hour at room temperature with 5% skim milk/TBS-T, andsubsequently probed with 1:1000 rabbit anti-BDNF (Abcam) or 1:1000rabbit anti-Ntrk2 (Abcam), overnight at 4° C. Anti-Rabbit-ECL secondary(GE, Mississauga, ON, Canada) at a concentration of 1:5000 was appliedfor 1 hour at room temperature, blots were briefly washed in TBS-T andTBS, then incubated with ECL substrate (Thermo-Scientific) for 5minutes. Exposures were performed using x-ray film (Thermo-Scientific),and the exposure times were 60, and 45 minutes for BDNF and Ntrk2respectively. Mouse brain was used as a positive control, and beta-actinas a loading control.

BDNF in the Human Uterus. pro-BDNF (35 kDa) was found to be the dominantform present whereas mBDNF was not detectable (FIG. 6A). A similaranalysis was conducted on endometrium from women with endometriosis andthe mBDNF form was overexpressed as compared to the other forms vs.controls.

Ntrk2 Expression in the Human Uterus. The truncated form of Ntrk2 wasfound to be the dominant isoform present whereas the full-length isoformwas low to non-detectable (FIG. 6B). A similar analysis was conducted onendometrium from women with endometriosis and the full-length (FL)variant of Ntrk2 was overexpressed compared to a truncated (T½) variantof Ntrk2 x vs. controls (FIG. 7).

Example 3—Determination of BDNF and Ntrk2 Expression by PCR

RNA from mouse, rat, human, pig, and horse was reverse transcribed usingthe iScript cDNA synthesis kit (Bio-Rad), according to kit protocol. PCRprimers were designed using human GenBank sequences for BDNF mRNA(NM_001143809.1) and Ntrk2 mRNA (NM_006180.3). Primers were designedagainst a 300 bp span within the coding region of the gene, and wheneverpossible were designed to span an intron. Primer3 software(http://frodo.wi.mit.edu/primer3/) was used for primer design andprimers were tested for hairpins, self-dimers, and hetero-dimers usingOligoAnalyzer 3.1(http://www.idtdna.com/analyzer/applications/oligoanalyzer/). Primersequences for BDNF were (Forward: GAGCTGAGCGTGTGTGACAG (SEQ ID NO:9),Reverse: CTTATGAATCGCCAGCCAAT (SEQ ID NO:10)), and for Ntrk2 (For ward:CAATTGTGGTTTGCCATCTG (SEQ ID NO:11), Reverse: TGCAAAATGCACAGTGAGGT (SEQID NO:12)). Primers were ordered from Mobix Laboratory (McMasterUniversity, Hamilton, ON, Canada), and diluted to a workingconcentration of 10 pmol/ml with DNase/RNase free water. cDNA for 3animals per group was pooled and used to isolate BDNF and Ntrk2transcripts. Real-Time PCR was performed in triplicate in a 10 mlreaction volume (2 ml pooled cDNA, 5 ml SYBR Green Master Mix (Qiagen),1 ml forward primer, 1 ml reverse primer, and 1 ml RNase/DNase freewater) using the capillary-based LightCycler (Roche Diagnostics, Laval,QC, Canada). The program was denaturation: 95° C. for 15 min;amplification: 55 cycles: 95° C. for 10 s, 56° C. for 5 s, 72° C. for 20s; melting curve: 70-95° C. at a rate of 0.1° C. per second.Amplification and melt curves were analyzed for each species using theLightCycler software (Roche Diagnostics). PCR products were collected,and sent for sequencing (Laboratory Services, University of Guelph).

Both primer pairs isolated specific products which were verified bysequencing in all species (human, mouse, rat, and horse).

Example 4

Several studies have indicated that estradiol (E₂) treatment alters theexpression of Ntrk2 and/or its ligand, BDNF, in neural tissue. Todetermine if BDNF expression was altered in the uterus, the followingstudy was conducted.

BDNF transcripts were measured in the murine uterus of ovariectomizedmice by Real Time PCR and relative expression quantified in micereceiving saline (Control (n=4)), estradiol primed then estradiol (E2(n=6)), estradiol primed then progesterone (P4 (n=6)), estradiol primedthen estradiol+progesterone (E₂+P₄ (n=6)), or estradiol primed thensaline (Saline (n=4).

Group Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 ControlSaline Saline Saline None None Saline Saline Saline Saline E2 E₂ E₂ E₂None None E₂ E₂ E₂ E₂ P4 E₂ E₂ E₂ None None P₄ P₄ P₄ P₄ E2&P4 E₂ E₂ E₂None None E₂ + P₄ E₂ + P₄ E₂ + P₄ E₂ + P₄ Saline E₂ E₂ E₂ None NoneSaline Saline Saline Saline

BDNF expression was significantly increased in the uterus ofovariectomized mice treated with E₂ and progesterone (P₄), an effectthat was further enhanced by co-treatment with E₂ and P₄ (FIG. 8).

Example 5—Biomarker Panel for Detecting or Monitoring Endometriosis

Study participants: Women (n=134) undergoing a laparoscopic procedure atMcMaster University Medical Centre for chronic pelvic pain were invitedto participate in this study. This study was approved by the Hamiltonintegrated Research Ethics Board, McMaster University (REB #12-083-T).Exclusion criteria included all women unable to consent, those under theage of 18, those who were pregnant, or those with a diagnosis ofadenomyosis. All women were asked to consent and complete aquestionnaire assessing demographics, menstrual cycle length, date oflast menstruation, and pelvic pain. During laparoscopic surgery, womenwere categorized as a case (n=96) or symptomatic control (n=25) by agynecological surgeon (NL) with extensive experience in the diagnosisand treatment of endometriosis. The stage of endometriosis was assignedduring surgery according to the revised American Society forReproductive Medicine (rASRM) classification system, as described inMedicine ASoR: Revised American Society for Reproductive Medicineclassification of endometriosis: 1996. FertSteril 1997, 67:817-21.Diagnosis was additionally confirmed through review of pathologyreports. Menstrual cycle stage was further confirmed by histopathology.

Participants who received hormone therapies within at least the 3 monthsbefore study enrollment were included as a separate subgroup of treatedcases (n=39) to determine the effect of ovarian suppression treatment oncirculating clinical markers. Thus, there were 57 untreated studyparticipants in the case group.

Sample Collection: Blood samples were collected from the cubital veininto serum and plasma separator tubes by a nurse at McMaster Universityhospital prior to surgery. Blood was placed on ice, transferred to thelaboratory, allowed to clot for 1 hour at 4° C., and centrifuged at3,000 rpm. Approximately 200 μL of plasma or serum was aliquoted into1.8 mL cryovials and frozen at −80° C. until required for analysis.Samples were stored in separate aliquots and only thawed once for eachassay to avoid repeat freeze/thaw cycles.

Quantification of Circulating Concentrations of Clinical Markers:Samples were thawed at room temperature and concentrations of eachprotein were quantified in duplicate using commercially available andexternally validated quantitative ELISA kits following themanufacturer's protocols. The clinical markers quantified include: brainderived neurotrophic factor (BDNF, Promega, Madison, Wis.), CA-125,VEGF, IL-1β, IL-6, RANTES, sICAM-1 (R&D Systems, Minneapolis, Minn.),z-alpha-glycoprotein (ZAG), leptin (Abnova, Walnut, Calif.), glycodelin(Biosery Diagnostics, Rostock, Germany), and SERPINE2 (Cloud-Clone Corp,Houston, Tex.). Optical densities were determined for each sample at awavelength of 450 nm.

The detection limits, and intra- and inter-assay coefficients ofvariation for each target protein measured were: BDNF (15.6 pg/ml, 2.2%and 8.6%), VEGF (9.0 pg/mL, 5.4% and 7.3%), IL-1β (0.033 pg/mL, <10% and<12%), IL-6 (0.7 pg/mL, 2.6% and 4.5%), RANTES (2.0 pg/mL, 2.4% and6.5%), ZAG (21 pg/mL, <10% and <15%), glycodelin (6 ng/mL, 8.3% and4.6%), sICAM-1 (0.096 ng/mL, 4.6% and 5.5%), leptin: (0.2 ng/mL, 5.9%and 5.6%), SERPINE2: (0.135 ng/mL, <10% and <12%), respectively.

Statistical Analysis: The proportions of study participants in eachgroup were compared by Chi-square. Circulating marker concentrationswere compared using t-test (cases vs. controls), or one-way ANOVA(comparisons between controls and disease stages) when data was normallydistributed. For non-normally distributed data, Mann-Whitney rank sumtest and Kruskal-Wallis one-way ANOVA on ranks were used. Statisticalanalyses were carried out using SigmaStat 3.5 software (Systat SoftwareInc., Chicago, Ill.). Data are presented as the mean±SD or median(25%-75% percentiles), as indicated. Results were consideredstatistically significant for p≤0.05.

To determine the effects of menstrual cycle phase on circulatingbiomarker concentrations, samples from controls were analyzed accordingto menstrual stage. If no significant differences were observed for abiomarker in either of these comparisons, subsequent analysis includedall controls as one group. Comparisons were then made between alluntreated cases and controls, untreated cases stratified by stage ofdisease (I-II vs. III-IV) and controls, and all cases stratified bytreatment (treated vs. untreated) and controls for each biomarker.Receiver operating characteristic (ROC) curves were generated and thearea under the curve (AUC) calculated. The sensitivity and specificityfor each marker was determined based on an optimal concentration cut-offvalue.

To evaluate potential benefits of combining clinical markers,Classification and Regression Tree (CART) analysis, a powerfulnonparametric statistical procedure for advanced predictive modeling(Predictive Modeler Software Suite version 7.0, Salford Systems, SanDiego, Calif., USA), was employed. Briefly, multiple potentialcontinuous predictor variables of a binary dependent variable (i.e.endometriosis or control) are inputted with the modeler examining allpossible dichotomous splits of subjects based on cut-off values ofpredictor variables. A decision-tree is created with different splitsthat best classify subjects (i.e. optimal sensitivity and specificity)with respect to the dependent variable. At the top of the decision-tree,two child nodes are created from a parent node (the “root” node”) withsubjects having a concentration of a particular biomarker greater than acut-off value being classified in one node, and subjects having aconcentration below a cut-off value for that particular biomarker beingclassified in the other node. The proportion of cases and controls bothabove and below the cut-off value are stated in each node. One (or both)of the child nodes then acts as a parent node for another set of childnodes with a different cut-off value of a different biomarkerdetermining how the subject population from the parent node is split.This process continues until terminal nodes are reached, which aremutually exclusive and exhaustive subgroups of the patient populationthat classify patients as either having the disease (cases=1) or not(controls=0). Sensitivities and specificities are then calculated basedon these classifications. Additionally, the tree-growing methodology canbe modified to avoid costly type I (false positive) or II (falsenegative) errors through adjustable misclassification penalties. Throughthese adjustments, a decision-tree optimized for sensitivity orspecificity can be created. This modification was used to create adecision-tree optimized for sensitivity. A biomarker was deemed suitablefor inclusion in CART analysis when ROC curves resulted in an AUC≥0.6.The minimum number of subjects in a terminal node was set to n=3.

Results

Patient Characteristics: All women included in this study (134)underwent laparoscopic surgery, from which 106 cases of endometriosisand 28 symptomatic controls where identified. Over all, 13 women wereexcluded from this study owing to a diagnosis of adenomyosis (casesn=10, controls n=3). The final study population constituted 121 women:96 cases and 25 controls as shown in Table 1.

TABLE 1 Patient characteristics of women with and without endometriosis.Characteristic Control (25) Cases (96) p Value Age (y), mean ± SD 34.3 ±8.3 33.6 ± 6.5 p = 0.722 Stage n (%) NA Minimal 1 0 (0) 8 (8) Mild 2 0(0) 7 (7) Moderate 3 0 (0) 10 (11) Severe 4 0 (0) 52 (54) Not available0 (0) 19 (20) Current Med n (%) p = 0.012 Hormonal contraceptives 4 (14)28 (27) Lupron 3 (11) 21 (20) NSAID 2 (7) 28 (27) Narcotic analgesic 1(4) 7 (7) none/other 18 (64) 34 (32) Menstrual Cycle Stage n (%)Menstrual 7 (28) 21 (22) p = 0.6  Proliferative 8 (32) 22 (23) Secretory4 (16) 22 (23) Unknown 6 (24) 31 (32) Duration of Bleeding, d Median 6(5-7) 6 (4-7) p = 0.198 (25%-75) Age at First Menstruation, y 13 (12-14)12 (11-13) p = 0.249 Median (25%-75) Ethnicity n (%) p = 0.095 Caucasian19 (76) 70 (73) Asian 4 (16) 8 (8) Black 0 (0) 5 (5) Aboriginal 1 (4) 0Unknown 1 (4) 13 (14) Occupational Status n (%) p = 0.329 Employed 19(76) 54 (56) Unemployed 1 (4) 11 (11) Other 1 (4) 5 (5) Unknown 4 (16)26 (27) Smoking Status p = 0.496 Yes 5 (40) 14 (15) No 20 (80) 77 (80)Unknown 0 4 (4) SD: standard deviation, y: years, d: days

The current study population consisted of cases with stage I (n=8),stage II (n=7), stage III (n=10), and stage IV (n=52). Disease stageinformation was unavailable for n=19 cases. Only current medication usediffered significantly (p=0.012) between cases and controls. Of the 96confirmed cases of endometriosis, n=39 (41%) received hormonal treatmentwithin three months prior to surgery, while n=57 (59%) were untreated.Of the 25 controls, n=7 (28%) reported use of OCP and non-steroidalanti-inflammatory drugs (NSAIDs) while n=18 (72%) denied medication use.The average age, ethnicity, occupational status, smoking status, age atfirst menstruation, median duration of bleeding, and menstrual cyclestage showed no significant differences between cases and controls.

Effect of Treatment on Peripheral Biomarkers: No significant differenceswere found when comparing treated and untreated controls for any of thebiomarkers tested. Similarly, there were no differences inconcentrations of any marker over the course of the menstrual cycle.Therefore, data from these groups were combined into a single controlgroup.

Biomarkers and Endometriosis: To determine the effect of endometriosison circulating biomarker concentrations, marker concentrations werecompared in samples from untreated cases (n=57) and all controls (n=25).Only glycodelin (p=0.041) and BDNF (p=0.0008) showed a significantdifference between untreated cases and controls (Table 2). Theconcentrations of ZAG approached but did not reach statisticalsignificance (p=0.086). ROC curves were generated for glycodelin, BDNF,ZAG, and CA-125 (FIG. 11A-D). The AUC for BDNF 0.73 (p<0.001) andglycodelin 0.70 (p=0.040) were statistically significant whereas the AUCfor ZAG 0.66 (p=0.085) and CA-125 0.47 (p=0.622) failed to reachsignificance (FIG. 11E). The sensitivity and specificity for each markerwas determined at cut-off values chosen to maximize marker accuracy(Table 2). Using a cut-off value of 944.6 pg/mL for BDNF yielded asensitivity of 68.3% (CI 55.0-79.7) and a specificity of 80.8% (CI60.7-93.5). For glycodelin, using a cut-off value of 19.8 ng/ml produceda sensitivity of 77.1% (CI 59.9-89.6) and a specificity of 58.3% (CI27.7-84.8). At a cut-off value of 91.6 pg/ml, ZAG achieved a sensitivityof 47.2% (CI 33.3-61.4) and a specificity of 100% (CI 73.5-100). At acut-off value of 14.9 U/mL CA125 achieved a sensitivity of 22.2% (CI12.0-35.6) and a specificity of 100% (CI 86.8-100).

TABLE 2 Concentrations of clinical markers in women with endometriosisvs. a control population. Marker Control (n = 25) Case (n = 96) p valueVEGF (pg/ml) 269.6 (96.6-350.1) 212.8 (124.1-339.2) 0.98 BDNF (pg/ml)629.7 (338.0-917.9) 1108 (663.8-1742) 0.0008 CA-125 (U/ml) 7.21(5.58-10.71) 7.30 (3.61-13.32) 0.63 IL-1β (pg/ml) 0.058 (0.033-0.10)0.052 (0.037-0.079) 0.64 IL-6 (pg/ml) 0.35 (0.06-1.41) 0.10 (0.06-0.80)0.58 RANTES (ng/ml) 33.4 (21.4-62.1) 37.4 (19.9-65.5) 0.57 sICAM (ng/ml)208.4 ± 46.0 218.1 ± 51.0 0.44 Glycodelin(ng/ml) 12.3 (5.2-31.4) 47.1(21.6-92.2) <0.001 ZAG (pg/ml) 50.4 (41.7-64.9) 73.7 (46.6-115.3) 0.085Leptin (ng/ml) 13.5 (8.7-22.7) 16.4 (8.3-25.2) 0.81 SERPIN2 (ng/ml) 16.1(13.4-24.8) 17.0 (14.3-20.7) 0.81 The data are presented as the median(25^(th)-75^(th) percentile) or the mean ± SD for normally distributeddata.

CART Analysis: Clinical markers with an ROC value greater than or equalto 0.60 were included in the analyses using CART software (SalfordSystems). The only markers investigated able to satisfy this criterionwere BDNF (AUC=0.73), glycodelin (AUC=0.70) and ZAG (AUC=0.66). CARTanalysis revealed that BDNF, glycodelin, and ZAG were able to form adecision-tree with a sensitivity of 76.9% and a specificity of 93.3% forthe diagnosis of endometriosis (FIG. 12). Given the high cost ofmisdiagnosing a patient with endometriosis as a control (falsenegative), the CART methodology was modified to create a decision-treeoptimized for sensitivity. This analysis produced a decision-tree ableto diagnose disease with a sensitivity of 89.2% and a specificity of70.0% (FIG. 13).

Markers and Stage of Disease: The effect of disease stage on peripheralmarker was determined through comparing biomarker concentrations ofuntreated cases with stage I-II disease (n=8), untreated cases withstage III-IV disease (n=44), and controls (n=25). BDNF and glycodelinwere the only markers to show significant variation in concentrationsbetween stages of disease. Median BDNF concentrations were significantly(p=0.0045) higher in stage I-II vs. III-IV stage disease (1147 vs. 1087pg/ml) and controls (629.7 pg/ml), suggesting that BDNF concentrationsare higher in early stage active disease (stage I-II). There was lessdifference in median glycodelin concentrations (p=0.41) between womenwith stage I-II disease and the control group (21.6 vs. 12.3 ng/ml),respectively, than between women with stage III-IV disease and thecontrol group. Thus, median glycodelin concentrations were significantly(p=0.027) lower in stage I-II vs stage III-IV disease (21.6 vs. 61.5ng/ml), suggesting that glycodelin concentrations rise with diseaseseverity.

Markers and Hormonal Treatment: The effect of hormonal treatment oncirculating levels of putative biomarkers in patients with endometriosiswas assessed. Of the clinical markers quantified, only BDNF (p<0.05) andglycodelin (p<0.001) demonstrated a significant difference betweentreated and untreated cases. BDNF concentrations in the plasma weresignificantly lower in treated than untreated cases (729.1 vs. 1097pg/ml). Similarly, median glycodelin concentrations were significantlylower in treated vs. untreated cases (8.37 vs. 47.1 ng/ml).

Discussion

In the present study, circulating concentrations of single biomarkersalone and in combination were evaluated for the diagnosis ofendometriosis in women undergoing laparoscopy for pelvic pain. Theresults revealed that circulating concentrations of BDNF and glycodelinwere significantly higher in women with endometriosis compared todisease free controls. Analysis by stage of disease revealed that thecirculating concentrations of BDNF were higher in stage I-II diseasecompared to stages whereas the reverse was true for glycodelin. Takentogether these results suggest that BDNF may be a better indicator ofactive disease, whereas glycodelin has value in identifying moreadvanced stages of endometriosis. CART analysis showed that thecombination of BDNF, glycodelin and ZAG yielded a sensitivity andspecificity of 89.2% and 70%, respectively.

In view of the lengthy delays in diagnosis and the invasive nature oflaparoscopy which itself is not perfect for the diagnosis ofendometriosis, these biomarkers could provide evidence in support ofclinical judgment for the empirical initiation of approved treatmentsfor endometriosis, thus allowing for quicker patient access to effectivemedical treatment and management. Further, this panel of markers maylead to the avoidance of unnecessary diagnostic laparoscopy in thosethat do not have endometriosis. Results of the present study alsorevealed that both BDNF and glycodelin concentrations were significantlylower in treated vs. untreated cases. For example, both BDNF andglycodelin had lower concentrations in women with endometriosis who weretreated with ovarian suppressing agents and, thus, these markers havevalue as indicators of treatment response, and thus, may replacelaparoscopy for this purpose.

BDNF, glycodelin, and ZAG produced acceptable ROCs for further study.Combining these three clinical markers in CART analysis yielded asensitivity and specificity of 76.9% and 93.3%, respectively. Thepositive predictive value (PPV) of this test was 96.2% with a negativepredictive value (NPV) of 65.1%. Optimizing the CART analysis to favorsensitivity over specificity we obtained adjusted sensitivity andspecificity values of 89.2% and 70.0%, respectively with a PPV of 86.6%and NPV of 75%.

In summary, BDNF and glycodelin were found to be superior to bothemerging (SERPINE2 and ZAG) and classical markers (CA-125, ZAG, VEGF,IL-6, IL-1β, RANTES, sICAM-1, and leptin) as single non-invasive markersfor the diagnosis of endometriosis in the present study population.Furthermore, combination of BDNF, glycodelin and ZAG in a panel produceda sensitivity and specificity that was superior to either marker alone.Both circulating BDNF and glycodelin concentrations have been positivelyassociated with pain and their concentrations declined with treatment.Therefore, the present clinical markers can be used to aid in clinicaldecisions to initiate effective medical treatment and management ofwomen with endometriosis, and BDNF and/or glycodelin can be used tomonitor patient response to treatment.

Example 6—Device for Detecting or Monitoring Endometriosis

Materials: BDNF antigen and anti-BDNF antibody, gold chloride (HAuCl),50% (w/w) glutaraldehyde, Potassium ferricyanide (FiCN, 99.0%), andcystamine were purchased from Sigma-Aldrich. Potassium chloride (KCl,≥99.0%), phosphate buffer solution (PBS, 1.0M, pH 7.4) was purchasedfrom Anachemia (Rouses Point, N.Y.). Pot Sulfuric acid (H2504, 98%),2-propanol (99.5%) were purchased from Caledon Laboratories (Georgetown,Ontario). Ethanol was purchased from Commercial Alcohols (Brampton, ON).Immobilized TCEP Disulfide Reducing Resin was purchased from ThermoScientific (Rockford, Ill.). Tris-(hydroxymethyl)aminomethane (tris,≥99.9%) was purchased from BioShop Canada (Burlington, ON). The hydrogenperoxide (H₂O₂) was purchased from Caledon. Milli-Q grade ultrapurewater (18.2 MΩ·cm) was used to prepare all solutions and for all washingsteps.

Device Fabrication: Two sets of devices were created: (1)electrochemistry on a linear surface (planar device); and (2) on aporous device (craft cut and (3-aminopropyl) triethoxysilane (APTES)solution-treated polymer). The first set of electrodes (CH Instruments,Austin, Tex.) were cleaned by polishing followed by sonification inethanol for two 5 minute intervals, DI water for 30 seconds, and thenair dried. For the second set, a clean commercially availablepolysterene (PS) substrate was plasma treated and incubated in a 10%APTES. The APTES acts as a self-assembled monolayer with amine affinityfor gold (Au). The substrate was covered with a self-adhesive vinylsheet. A Robo Pro CE5000-40-CRP vinyl cutter equipped with a CB09UAsupersteel blade was used with Adobe Illustrator [v.16.0.3] CAD modelingsoftware to create the desired shapes. The masked surface is attached tothe substrate and incubated in Au nanoparticle (NP) solution overnightto form a seed layer. Electroless deposition was performed on theplanted seed layer by immersion into chloroauric acid (HAuCl4) followedby an injection of hydrogen peroxide (H₂O₂) for 2 minutes and then theAu-treated substrate was rinsed with water. Following the electroless Audeposition process, the vinyl mask was removed with tweezers and rinsedwith DI water. The devices were heated for 3 minutes at 160° C. in orderto be shrunk and rinsed again.

Deposition of Immunosensor Probes for Protein Detection: For both planarand porous electrode experiments, a self-assembled monolayer (SAM) wascreated as the primary linker of the detection device by applying a 2 Mcystamine solution at room temperature overnight. An optimization stepwas performed to determine the effectiveness of increasingconcentrations of cystamine. A 2 M cystamine solution yielded the bestresults and thus was used for all subsequent procedures. Before applyingthe cystamine solution, a 1 hour reduction was performed with TCEPdisulfide reduction solution to lyse the sulfide bond in the cystaminemolecule, which is required for the immobilization of the secondarylinker. After being rinsed with DI water, the SAM was introduced to thesecondary linker, 2.5% glutaraldehyde in water, for 1 hour and rinsedagain.

To test the ability of the devices to detect BDNF, 10 ug/mLanti-BDNF-antibody in phosphate buffered saline (PBS) was incubated withthe device at room temperature for 1 hour. After being washed with PBStwice for 5 minutes, 5% (w/v) bovine serum albumin in PBS was applied tothe sensor surface in order the block the unreacted aldehyde groups sothat they do not interfere with the electrochemical signal. The sensorswere washed 3 times with PBS. Increasing concentrations of BDNF (0.5, 1,and 2 ng/ml) in PBS were applied to the immunosensors for 40 minutes at37° C. and washed with PBS and water before electrochemical measurementswere assessed.

Electrochemical Measurements: All electrochemical experiments wereperformed using a CH1 660D potentiostat (CH Instruments, Austin, Tex.)connected to a 3-electrode arrangement consisting of a referenceelectrode Ag/AfCl (1.0 M KCl) and a counter electrode platinum wire. Theporous working electrode was created with three 2×2 mm square electrodesper device connected with thin gold lines to a contact pad which isattached to metal clip. The planar working electrode is alreadycustomized with a built in clip. The surface areas of the porous andplanar electrodes were calculated using cyclic voltammetry (CV) in a0.05 M H2SO4 solution by integrating the Au reduction peaks of thecyclic Voltammogram and dividing that reduction charge by the surfacecharge density. The surface charge density of the Au monolayer wasexperimentally determined to be 386 uC/cm.

Protein Detection and SEM Images: Electrochemical signals were measuredin an electrochemical solution containing 2.5 mM K3[Fe(CN)6], 2.5 mMK2[Fe(CN)6], 0.1 M KCl, and 10 mM phosphate buffer solution (PBS).Cyclic voltammetry (CV) was obtained with a scan rate of 100 mV/s, anddifferential pulse voltammetry (DPV) signals were obtained with apotential step of 5 mV, pulse amplitude of 50 mV, pulse with 50 ms, anda pulse period of 100 ms (as described by Woo et al. 2014). Signalpercentage changes corresponding to target protein binding to theantibody were determined by calculating the difference between theaverage current before and after adding the target analyte. Threedifferential pulse voltammograms were performed per concentration toensure accuracy of the reading. SEM images of planar and porous goldelectrodes were obtained using a JEOL JSM-70005 scanning electronmicroscope with an accelerating voltage of 2 kV, working distance of 6mm, and low probe current.

Clinical samples: To demonstrate the ability of the device to detectplasma BDNF concentrations and discriminate between cases and controls,plasma samples were collected and utilized. Briefly, five samples thathad circulating concentrations of BDNF above 1.0 ng/ml (cases) and fivesamples in which the concentrations of BDNF were previously shown to beless than 0.6 ng/ml (controls) were randomly selected from the archiveand coded. Each sample was tested in triplicate by a study team member(MB) blinded to prior study results. Once all the scans were completedthe code was broken and results assigned to the case or control groupbased on results from the original study and data from the currentstudy.

Results and Discussion

The process for making the porous APTES-treated polystyrene-basedelectrode is shown in FIG. 16. The template for the seed layer wasdesigned using CAD program and self-adhesive vinyl was cut and placed asa mask. A seed layer of a 12 nm gold nanoparticles was then used to fillthe pores with a gold film. The gold film was then activated with theaddition of hydrogen peroxide (H₂O₂) and gold chloride (HAuCl₄). Thiselectroless method does not require photolithography, which isadvantageous for cost efficient multiplexing of chips on a singlesubstrate. The chips were then shrunk thermally in the oven at 160degrees C. for 3 minutes. It is the shrinking process that causes heightvariations in the electrode surface, therefore creating pores. Theporous electrodes demonstrated an increased limit of detection comparedto the planar electrodes because it increases surface area, and thus,sensitivity of the biosensors through its textured surface (see FIG.17).

In order to functionalize the electrodes as immunosensors, aself-assembled monolayer of cystamine was immobilized on the goldsurface through a thiol bond because thiols have a high affinity formetals. Additionally, its amine functional group provides the abilityfor the bifunctional linker, glutaraldehyde, to attach. Gluteraldehydeprovides a ketone functional group that is capable of attaching abiomarker-specific reactant, such as an antibody, for detecting a targetanalyte (e.g. BDNF). Adding an aqueous solution of each chemical overthe chips forms these linkers. Moreover, the anti-BDNF monoclonalantibody is the probe on the electrode surface that will specificallyattach to its target analyte, BDNF protein. Antibodies circulate inplasma as an immunological response system that detect and attach toforeign entities. The variable region of the antibody polypeptide chainrecognize and attach to its corresponding antigen, BDNF. Monoclonalantibodies are ideal because they are highly specific and are exclusiveto one epitope.

In order to correlate the concentration of BDNF protein attached to theantibody probes, a voltage is applied to generate an electrochemicalsignal from the working electrode, which is comprised of the antibodyprobes. The working electrode is surrounded by a redox reporter,[Fe(CN)₆]^(3−/4−), which produces a faradaic current through interfacialelectron transfer. A comparison of the electrochemical signal with andwithout protein analyte was performed and illustrated a decrease insignal in the presence of protein since the protein blocks the redoxreporter from accessing the electrode surface. Less electron transferequals less current produced, therefore a smaller current signal ismeasured. These signals are measured using a differential pulsevoltammogram, which illustrated the reduction peak as interfacialelectron transfer occurs (FIG. 19). Using known concentrations of BDNFprotein in PBS (1×), the mean difference in current change wascalculated for increasing concentrations (FIG. 18). This information isused to determine BDNF concentrations in blood samples by calibratingthe detection system with the known concentration standards between 1ng/mL and 4 ng/mL.

CONCLUSION

Through the use of a three electrode system, deciphering positiveprotein-antibody binding correlates to the positive identification ofdisease progression, specifically, the amount of adhesions in anendometriosis patient. Smooth or porous electrodes may be used.Alteration of the electrode surface morphology has demonstrated anincrease in sensitivity regarding a probe vs. BDNF protein signal changeboth in PBS and patient plasma. In either case, electrodes have beensuccessfully utilized to detect a target analyte, such as BDNF, and maybe utilized to detect other endometriosis biomarkers as well, eitherseparately or in a multiplexed platform.

1-12. (canceled)
 13. A method of diagnosing endometriosis in a human,the method comprising: detecting an expression level of circulating BDNFin a biological fluid sample taken from the human; comparing thecirculating BDNF expression level to a BDNF control level to determineif the circulating BDNF level is elevated as compared to the controllevel, wherein the control level is an expression level of BDNF in ahuman without endometriosis; diagnosing the human with endometriosiswhen the circulating BDNF expression level is elevated by at least 10%as compared to the BDNF control level; and further diagnosing a stage ofthe endometriosis if present, wherein elevation of the circulating BDNFexpression level by 30% to 50% as compared to the BDNF control level isindicative of stage 1 or stage 2 endometriosis.
 14. The method of claim13, wherein the biological sample is selected from the group consistingof blood, serum, plasma, urine and peritoneal fluid.
 15. The method ofclaim 13, further comprising determining the expression level of CA-125in the biological sample from the human and comparing the CA-125expression level to a CA-125 control level, wherein the control level isan expression level of CA-125 in a human without endometriosis.
 16. Themethod of claim 13, wherein the control level of BDNF is 100-500 pg/ml.17. The method of claim 13, wherein the BDNF is total plasma BDNF. 18.The method of claim 13, wherein the determining step comprisescontacting the biological fluid sample with an anti-BDNF antibody anddetecting binding between the BDNF from the biological fluid sample andthe anti-BDNF antibody, wherein the anti-BDNF antibody is selected fromthe group consisting of a polyclonal antibody, a monoclonal antibody,and an antigen-binding fragment.
 19. A method of diagnosingendometriosis in a human with pelvic pain, the method comprising:detecting an expression level of circulating BDNF in a biological fluidsample taken from the human with pelvic pain; comparing the circulatingBDNF expression level to a BDNF control level to determine if thecirculating BDNF level is elevated as compared to the control level,wherein the control level is an expression level of BDNF in a humanwithout endometriosis; and diagnosing the human with endometriosis whenthe circulating BDNF expression level is elevated as compared to theBDNF control level.
 20. The method of claim 19, wherein the biologicalsample is selected from the group consisting of blood, serum, plasma,urine and peritoneal fluid.
 21. The method of claim 19, wherein thedetermining step comprises contacting the biological fluid sample withan anti-BDNF antibody and detecting binding between the BDNF from thebiological fluid sample and the anti-BDNF antibody, wherein theanti-BDNF antibody is selected from the group consisting of a polyclonalantibody, a monoclonal antibody, and an antigen-binding fragment. 22.The method of claim 19, further comprising determining the expressionlevel of cancer antigen CA-125 in the biological sample from the humanwith pelvic pain and comparing the CA-125 expression level to a CA-125control level, wherein the control level is an expression level ofCA-125 in a human without endometriosis.
 23. A method of diagnosingendometriosis in a human, the method comprising: detecting theexpression level of circulating BDNF and CA-125 in a biological fluidsample taken from the human; comparing the circulating expression levelsof BDNF and CA-125 to control levels of BDNF and CA-125 to determine ifthe circulating BDNF and CA-125 levels are elevated as compared to thecontrol levels, wherein the control levels are expression levels of eachof BDNF and CA-125 in a human without endometriosis; and diagnosing thehuman with endometriosis when one or both of the circulating BDNF andCA-125 expression levels are elevated as compared to the BDNF and CA-125control levels.
 24. The method of claim 23, further comprising:diagnosing the human with endometriosis when the circulating BDNFexpression level is elevated by at least 10% as compared to the BDNFcontrol level; and further diagnosing a stage of the endometriosis,wherein elevation of the circulating BDNF expression level by 30% to 50%as compared to the BDNF control level is indicative of stage 1 or stage2 endometriosis.
 25. The method of claim 23, wherein the biologicalsample is selected from the group consisting of blood, serum, plasma,urine and peritoneal fluid.
 26. The method of claim 23, wherein thedetermining step comprises contacting the biological fluid sample withan anti-BDNF antibody and an anti-CA-125 antibody, and detecting bindingbetween the BDNF and the anti-BDNF antibody and between the CA-125 fromthe biological fluid sample and the anti-CA-125 antibody, wherein theanti-BDNF and anti-CA-125 antibodies are selected from the groupconsisting of a polyclonal antibody, a monoclonal antibody, and anantigen-binding fragment.
 27. The method of claim 23, wherein the humanhas pelvic pain.